Transgenic Ficus, Method for Producing Same and Use Thereof

ABSTRACT

The present invention relates to transgenic  Ficus  plants, particularly  Ficus carica  (fig tree), to a method for producing same, and to  Ficus  plants, plant materials and plant products produced by or from such genetically modified plant material. More specifically, the present invention relates to transgenic  Ficus carica  plants and use thereof for producing trees having improved agricultural traits and for the production of foreign proteins and edible vaccines.

FIELD OF THE INVENTION

The present invention relates to genetically modified Ficus plants,particularly Ficus carica (fig tree), to a method for producing same,and to Ficus plants, plant materials and plant products produced by orfrom such genetically modified plant material. More specifically, thepresent invention relates to a method for efficient transformation andregeneration of Ficus carica plants and use thereof for producing treeshaving improved agricultural traits and for the production of foreignproteins and edible vaccines.

BACKGROUND OF THE INVENTION

The genus Ficus of the Moraceae family is a large genus including about30 species. Ficus species are commonly used in gardening, in private aswell as in public gardens. Most species bear fruit; however, in severalspecies, fruit is considered a problem since the fruit are in-edible andfalling fruit litter sidewalks and other pathways. The edible fruit, onthe other hand, are highly desired as they are tasty and nutritional.

Fig trees are one of the earliest fruit bearing trees cultivated. Ficuscarica L. (Moraceae), the well-known fig of commerce is indigenous toareas from Asiatic Turkey to North India, and natural varieties arecultivated in most of the Mediterranean countries. The fig is well knownfor its nutritive value, and is consumed fresh or as dried fruitworldwide. The fig fruits are also known for their mild laxativeactivity and high alkalinity, and substances derived therefrom are usedin various drug preparations. Other parts of fig trees have also beenshown to have a commercial value.

For example, U.S. Pat. No. 6,235,860 has recently disclosed the use ofFicus carica as a source for natural rubber.

U.S. Pat. No. 5,494,669 relates to a skin disorder known aspseudofolliculitis barbae (PFB), and more particularly to a preparationfor treating PFB. The preparation disclosed is a topical solutioncomprising a mixture of alophatic alcohol, liquid aloe, liquid camphor,and the soluble materials of the fresh fig leaves of Ficus carica.Similar compositions are disclosed as a massage composition (U.S. Pat.No. 4,582,706).

U.S. Pat. No. 6,184,193 discloses a shrinkage prevention agent for wetwashing of clothing that would conventionally have been dry-cleaned. Theshrinkage prevention agent disclosed comprises a steam or vacuumdry-distilled liquid of bark, leaves, stems or flowers of two or moreplants, including Ficus carica.

Traditional breeding methods of Ficus carica require a long-term effortfor improving traits of fig trees. Among the Ficus carica some treesbear only female synconium and others, named caprifig, bear both maleand female flowers. True fruits can be produced only on tress bearingfemale synconium and cross-pollination is mediated by a specific wasp.Thus, traits donated by the stamens are hard to track.

The application of genetic engineering techniques to stably incorporatehomologous and/or heterologous genetic material into woody species,including fruit trees, offers the potential of obtaining improvedplanting stocks for private and public gardens and for agricultural usein a short period of time compared to those developed using traditionalbreeding techniques. In addition, efficient transformation methods canbe used for the production of heterologous polypeptides havingnutritional and/or pharmaceutical value, including edible vaccines.

Plants have the capacity to express foreign genes from a wide range ofsources, including viral, bacterial, fungal, insect, animal, and otherplant species. In single-copy nuclear transgenics, foreign protein inexcess of 1% of total protein is often achieved (Hiatt et al., 1989.Nature 342:76-78). Further, assembly and processing of complex animalproteins in plants is possible, e.g., human serum albumin (Sijmons etal., 1990. Bio/Technology 8:217-220) and secretory antibodies (Ma etal., 1995. Science 268:716-719.). Expression of correctly processedavidin in corn seed at a level of 2% of the total soluble protein wasalso reported (Hood et al., 1997. Mol. Breeding. 3:291-306). It has beenestimated that the cost of recombinant protein production in plants(assuming the foreign protein is 10% of total protein) can be 10 to 50times less than in E. coli by fermentation (Kusnadi et al., 1997.Biotechnol. Bioeng. 56:473-484). Many plant species are now amenable togene transfer, and numerous number of patents disclose different plantspecies capable of expressing foreign proteins; U.S. Pat. No. 6,392,121discloses a system for gene amplification based on plant viral geneticelements that can be used to increase the production of foreign proteinswithin the plant.

Vaccines are administered to humans and animals to induce their immunesystems to produce antibodies against viruses, bacteria, and other typesof pathogenic organisms. In the economically advanced countries of theworld, vaccines have brought many diseases under control. In particular,many viral diseases are now prevented due to the development ofimmunization programs. However, many vaccines for various diseasesincluding poliomyelitis, measles, mumps, rabies, foot and mouth, andhepatitis B are still too expensive for the lesser-developed countriesto provide to their large human and animal populations. Because ofsimplicity of delivery of vaccines by oral delivery, there is greatcurrent interest in discovering new oral vaccine technologies.Appropriately delivered oral immunogens can stimulate both humoral andcellular immunity and have the potential to provide cost-effective, safevaccines for use in developing countries or inner cities wherelarge-scale parenteral immunization is not practical or extremelydifficult to implement. Such vaccines may be based upon bacterial orviral vector systems expressing protective epitopes from diversepathogens (multivalent vaccines) or may be based upon purified antigensdelivered singularly or in combination with relevant antigens or otherpathogens. Several methods for using transgenic plants for oralimmunization have been disclosed, for example International PatentApplication WO 99/54452; U.S. Pat. Nos. 5,484,719; 5,612,487: 5,914,123;6,034,320; 6,084,152; 6,194,123; 6,395,964 and 6,444,805.

The overall efficiency of techniques for genetically modifying plantsdepends upon the efficiency of the transformation technique(s) used tostably incorporate the required genetic material into plant cells ortissues, and the regeneration technique(s) used to produce viable plantsfrom transformed cells.

Within Ficus species, there were some reports on regeneration andorganogenesis from callus and explants. Ficus religiosa plants have beenregenerated from callus of stem segments (Jaiswal and Narayan, 1985.Plant Cell Reports 4:256-258) and leaf callus (Narayan and Jaiswal, 1986Ind. J. Exper. Bio1.24:193-194). Regeneration of Ficus lyrata plantsfrom the axillary buds of a mature tree, and the formation of buds fromleaf explants were also reported (Deshpande et al., 1998. Plant CellReports 17:571-573). However, for in vitro work with figs, plantregeneration has been restricted to the use of single shoot tips andapical buds.

Recently, Yakushiji et al. (J. of Hort. Sci. & Biotech. 2003, 78,874-878) reported a method for the induction of organogenesis from leafexplants of Ficus carica using phloroglucinol (PG). However, by thismethod the frequency obtained for adventitious bud differentiation fromleaf fragments was relatively low, and no adventitious buds wereobserved without PG. Moreover, regeneration was obtained only withnon-transformed leaf segments. Although methods for transformation ofwoody plants have been reported (for example U.S. Pat. No. 6,255,559),no method for the introduction of isolated genetic material into Ficusspecies was hitherto disclosed.

Thus, there is a recognized need for, and it would be highlyadvantageous to have efficient methods for both transformation andregeneration of commercially valuable Ficus species, specifically figtrees, providing de novo origination of plant material from transformedcells and development of the genetically modified plant material toproduce genetically modified plants.

SUMMARY OF THE INVENTION

The present invention relates to transgenic Ficus species, particularlyto the commercially valuable fig tree Ficus carica, comprising exogenouspolynucleotides homologous or heterologous to the target Ficus genomeand to transgenic Ficus species capable of expressing exogenouspolynucleotides. The present invention further relates to methods forefficient transformation and regeneration of Ficus species. The presentinvention also relates to genetically modified Ficus species havingimproved agronomical traits, useful, inter alia, for production offoreign proteins and production of edible vaccines.

According to one aspect, the present invention provides transgenic Ficusspecies, specifically Ficus carica, comprising a genetic constructcomprising at least one exogenous polynucleotide.

According to one embodiment, the genetic construct further comprises oneor more regulatory elements to confer functional expression of theexogenous polynucleotide in the target Ficus plant. As used herein, theterm “regulatory element” refers to a non-coding polynucleotideregulating the expression of the exogenous polynucleotide. Regulatoryelements include, for example, constitutive, inducible ortissue-specific promoters; enhancer elements; termination elements;transposable elements; and post-transcriptional regulatory elements. Thepractice of the present invention is not bound to a specific constructand any construct suitable for plant transformation as is known to aperson skilled in the art can be used. Introduction of the geneticmaterial into the Ficus plant can be performed by any suitabletransformation method, including, but not limited to,Agrobacterium-mediated introduction, protoplast fusion, viral-mediatedtransformation, high velocity projectile introduction, electroporation,injection into reproductive organs, and injection into immature embryos.

According to yet another embodiment, the genetic construct according tothe present invention further comprises a selection marker. Selectionmarkers are well known in the art, and the selection technique may varydepending upon the selection marker used. According to one embodiment,the selection marker is a gene inducing antibiotic resistance, enablingthe survival of the transgenic Ficus plants in a medium containing theantibiotic as a selection agent. According to another embodiment, theselection marker is a reporter gene. The reporter gene can encode afluorescent protein, a chemiluminescent protein, a protein having adetectable enzymatic activity and the like, as is known to a personskilled in the art.

According to yet another embodiment, the selection marker is a herbicideresistance gene, enabling the survival of the transgenic Ficus plants ina medium containing the herbicide as a selection agent.

According to further embodiment the exogenous polynucleotide is anexogenous polynucleotide selected from the group consisting ofpolynucleotides homologous to the Ficus genome and polynucleotidesheterologous to the Ficus genome. The polynucleotide may be selectedfrom a polynucleotide encoding a polypeptide or a functional portion ofa polypeptide, a polynucleotide encoding a regulatory factor, such as atranscription factor, a non-coding polynucleotide such as a regulatorypolynucleotide, and antisense polynucleotide that inhibit expression ofa specified polypeptide.

According to one embodiment, the transgenic Ficus plant comprises apolynucleotide conferring a desirable agronomical trait selected fromthe group consisting of, but not limited to, insect tolerance, diseaseresistance, herbicide tolerance, rooting ability, cold tolerance,drought tolerance, salinity tolerance, modified growth rate, modifiedfruit development, fruit with better appearance, fruit with bettertaste, fruit with better storage ability, fruit with better shelf life,and improved dried fruit.

According another embodiment, the exogenous polynucleotide transformedinto the Ficus plant encodes a foreign protein. As used herein, the term“foreign protein” refers to a polypeptide or a protein not naturallypresent in the Ficus species to be transformed. According to oneembodiment, the foreign protein is expressed in an edible part of theplant, specifically in the edible fruit of Ficus carica. As used herein,an edible fig fruit is defined as the multiple fruit developed from thecomplex fig inflorescence called synconium. The multiple fruit consistof fleshy receptacle surrounding by diminutive drupe that form from eachpistillate flower. According to another embodiment, the foreign proteinis expressed in the latex serum produced by the Ficus tree. The foreignprotein may be utilized while maintained within the transgenic planttissue, for example by consumption of transgenic edible Ficus fruit orby employing the latex serum. Alternatively, the foreign protein may beextracted from the transgenic Ficus plants.

According to one embodiment, the Ficus plant is the fig tree Ficuscarica and the exogenous polynucleotide encodes a polypeptidecontributing to the nutritional composition of the Ficus carica ediblefruit.

According to another embodiment, the Ficus plant is the fig tree Ficuscarica and the exogenous polynucleotide encodes a protein contributingto the therapeutic value of the Ficus carica edible fruit. Preferably,the protein is selected from the group consisting of an anti-canceragent, an anti oxidant and a protein eliciting an immunogenic responsein a mammal.

According to one embodiment, the present invention provides a transgenicFicus carica plant producing fruit comprising at least one exogenouspolynucleotide expressing one of the group consisting of a polypeptide,a functional portion of a polypeptide, a peptide, a protein and a fusionprotein eliciting immunogenic response in a mammal, wherein theexpression is at a level such that upon oral administration to a mammal,an immunogenic response is observed, thus forming an edible vaccine. Thevaccine can be any vaccine as is known to a person skilled in the art,including but not limited to a vaccine for the treatment of hepatitis,malaria and cholera.

Pollen and ovules from the transgenic Ficus plants; the seeds producedfrom same and the plants grown from the seeds; edible fruit produced bythe genetically modified plants; and plants regenerated from tissuecultures regenerated from the genetically modified plants of the presentinvention are also encompassed within the scope of the presentinvention.

According to yet another embodiment, the present invention provides atissue culture regenerated from the transgenic Ficus plants of thepresent invention, wherein the tissue culture comprises cells orprotoplasts from a transgenic tissue selected from the group consistingof leaves, pollen, embryos, roots, root tips, anthers, flowers, fruitand seeds.

According to another aspect, the present invention provides a method forproducing transgenic Ficus species comprising:

a) transforming a genetic construct comprising at least one exogenouspolynucleotide into at least one Ficus explant to form a putativelytransformed explant;

b) inducing the regeneration of at least one adventitious shoot on theputatively transformed explant to obtain putatively transformedadventitious shoot;

c) selecting a transformed adventitious shoot; and

d) culturing the transformed adventitious shoot to form a transgenicFicus plantlet.

According to one embodiment, the explants are obtained from an in vitroFicus culture. According to one currently preferred embodiment, the invitro culture is prepared by horizontally placing shoot tips of Ficus ona solid propagation medium enabling shoot growth and leaf development.According to one embodiment, the propagation medium comprises at leastone cytokinin, at least one auxin and optionally at least onegibberellin.

Techniques for transforming genetic constructs into the genome of targetplants are well known in the art and include Agrobacterium-mediatedintroduction, electroporation, protoplast fusion, injection intoreproductive organs, injection into immature embryos, high velocityprojectile introduction, and the like. The choice of technique willdepend upon the Ficus species to be transformed. Targets for theintroduction of the genetic constructs of the present invention includetissues, such as leaf tissues, disseminated cells, protoplasts, seeds,embryos, meristematic regions, cotyledons, hypocotyls, and the like.According to one embodiment target plant material for transformationaccording to the methods of the present invention include explantsobtained from in vitro cultures prepared as described above. The term“transformation” refers to the introduction of an isolatedpolynucleotide into a plant cell, either in culture or into the tissuesof a plant.

According to one embodiment, transforming the explants is performed byco-culturing the explants with Agrobacterium culture harboring thegenetic construct comprising the desired genetic material. Thepolynucleotide may integrate into the host cell genome (“stabletransformation”) or be expressed without such integration (“transienttransformation”). According to one embodiment, co-culturing is performedwith explants placed on a solid medium, abaxial side up. As used herein,the term “abaxial” refers to a surface facing away from the plant axis,as the lower side of a leaf; the term “adaxial” refers to a surfacefacing towards the plant axis, as the upper side of a leaf.

The present invention now discloses that surprisingly, transformation ishighly dependent on the dorsoventral orientation of the explants.

Agrobacterium-mediated transformation occurs only when co-culturing isperformed on a solid medium with the explants placed with their abaxialside up. No transformation is observed when the explants are placedadaxial side up.

According to one embodiment, the explants are wounded before culturingwith Agrobacterium. According to one currently preferred embodiment, theAgrobacterium strain used is Agrobacterium tumefaciens. According toanother embodiment, the co-culturing medium is a solid medium comprisingat least one auxin and at least one cytokinin.

According to one embodiment, the Ficus explants taken for transformationare leaf explants. Fully developed leaves are excised from the in vitroshoot culture and taken for transformation.

The putatively transformed explants are transferred to conditionsinducing the formation of adventitious shoots. According to oneembodiment, the induction of adventitious shoots is obtained by placingthe putatively transformed explants, adaxial side up, on a solidregeneration medium under low light intensity; transferring the explantsto normal light intensity; and subsequently transferring the explants toa propagation medium under normal light intensity. As used herein “lowlight intensity” refers to light intensity from about 1 to about 5μmol/m² s, and “normal light intensity” refers to light intensity fromabout 10 to about 60 μmol/m² s. According to one embodiment, theregeneration medium comprises at least one auxin, at least one cytokininand at least one carbon source. According to another embodiment, theregeneration and the propagation media further comprise at least oneselection agent. According to one currently preferred embodiment theselection agent is an antibiotic.

The frequency of shoot regeneration obtained by the method of thepresent invention, for wild type as well as transgenic Ficus species, isat least 80%, preferably 90%, more preferably 100%, with regenerationefficacy of at least 3 shoots per explant, preferably 5 shoots perexplant, more preferably 10 shoots per explant. As used herein, the term“regeneration frequency” refers to the percentage of explants formingadventitious shoots. The term “regeneration efficacy” as used hereinrefers to the number of shoots regenerating on one explant. According toone currently preferred embodiment, the explants are leaf explants.After the development of adventitious shoots, only the transformedshoots are selected.

According to one embodiment the genetic construct of the presentinvention further comprises a selection marker conferring antibioticresistance. The transformation method using a selection markercomprises:

a) exposing the putatively transformed explants to a regeneration mediumcomprising a first concentration of a first antibiotic and a secondantibiotic, to obtain surviving regenerated shoots;

b) transferring the surviving shoots to a propagation medium comprisinga second concentration of the first antibiotic and the secondantibiotic;

c) repeating step (b); and

d) transferring the surviving shoots of step (c) to the propagationmedium comprising the second antibiotic.

According to one embodiment, the regeneration and propagation media arethe media described herein above.

According to one embodiment, the second concentration of the firstantibiotic is greater than its first concentration, and theconcentration of the second antibiotic is constant. According to onecurrently preferred embodiment, the first antibiotic is kanamycin andthe second antibiotic is ticarcillin.

Following selection of transformed shoots, the shoots are transferred toa rooting medium and roots are generated using techniques that are wellknown in the art. Rooted transgenic shoots, designated herein astransgenic plantlets, may be grown to mature transgenic Ficus plants.The plantlets include the genetic material introduced using the geneticconstruct according to the present invention. The products obtained frommature transgenic plants, including fruit, timber, latex serum, woodpulp, fuel wood, and the like, also contain the genetic modification.

The transformation and regeneration methods of the present inventionprovide means for the introduction of at least one exogenouspolynucleotide into plants of the genus Ficus, specifically Ficuscarica. The polynucleotide can be, for example, new gene, an additionalcopy of an existent gene, or a regulatory element. According to oneembodiment, the transformation and regeneration methods of the presentinvention are utilized to introduce genetic material that confersdesirable agronomical traits, selected from the group consisting of, butnot limited to, insect tolerance, disease resistance, herbicidetolerance, rooting ability, cold tolerance, drought tolerance, salinitytolerance, modified growth rate, modified fruit development, fruit withbetter appearance, fruit with better taste, fruit with better storageability, fruit with better shelf life, and improved dried fruit.According to this embodiment, the genetic material introduced may behomologous or heterologous to the genome of the target plant.

According to another embodiment, the transformation and regenerationmethods of the present invention are utilized to introduce into Ficusplants at least one exogenous polynucleotide capable of expressing aforeign protein. Expression of the foreign protein can be constitutiveor dependent upon induction, tissue specific or general. The Foreignprotein can be extracted from the plant tissue for further purificationand use, or can be used while maintained in the Ficus plant tissue.According to one embodiment, the foreign protein is expressed in anedible part of the plant, specifically in the edible fruit of Ficuscarica.

According to one embodiment, the expressed foreign protein contributesto the nutritional composition and value of the fruit.

According to another embodiment, the expressed foreign proteincontributes to the therapeutic value of the fruit. According to onecurrently preferred embodiment, the foreign protein is selected from thegroup consisting of an anti cancer agent, an anti oxidant and a proteineliciting an immunogenic response in a mammal.

According to one currently preferred embodiment, the transformation andregeneration methods of the present invention are utilized for theproduction of a transgenic Ficus carica producing fruit comprisingprotein eliciting immunogenic response in a mammal, wherein the proteinis expressed in the plant at a level such that upon oral administrationto a mammal, an immunogenic response is observed, thus forming an ediblevaccine. The vaccine can be any vaccine as is known to a person skilledin the art, including but not limited to a vaccine for the treatment ofhepatitis, malaria and cholera.

The present invention is better explained by the description, figuresand claims that follow.

BRIEF DESCRIPTION OF THE FIGURES

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-b describe the effect of growth regulator concentration andlight conditions on shoot regeneration of cv. Brown Turkey. Leafexplants were cultured with the abaxial side up following (A) one weeklow intensity light, (B) two weeks dark, one week low intensity light,and subsequently transferred to normal light. Vertical bars indicatestandard error (±S.E.).

FIGS. 2 a-c show light microscopy observation of shoot organogenesis inthe fig tree. Leaf explants of cv. Brown Turkey were cultivated onmedium with IBA (2 mg/l) and TDZ (2 mg/l). (A) After 15 days—the firstmeristematic domes appeared on the adaxial surface of the explant.Bar=100 μm. (B) After 21 days—adventitious buds and leaf primordia onthe adaxial epidermis. Bar=200 μm. (C) After 28 days—adventitious shootswith differentiated apical and axillary meristems and developing leaves.Bar=100 μm; C— callus, CD-cell division, DL-developing leaves,E-epidermis, M-meristem, VS-vascular system.

FIG. 3 shows the influence of sucrose concentration and leaf surfaceposition on adventitious shoot regeneration. Leaf explants of cv. BrownTurkey were cultured on medium with TDZ (2 mg l⁻¹) and IBA (2 mg l⁻¹).Vertical bars indicate standard error (±S.E.).

FIGS. 4 a-b show formation of adventitious shoot in fig cv. BrownTurkey. Stereomicroscopy observation of the leaf explants cultured 4weeks on regeneration medium (A) with adaxial side up (0.63×0.8) and (B)with abaxial side up (0.63×1.25).

FIGS. 5 a-c describe rooting and acclimatization of plants from cv.Brown Turkey. (A) In vitro rooted plant (bar 1 cm). (B) Plants inrooting cylinders. (C) Potted plant in the greenhouse one month afteracclimatization.

FIG. 6 describes the sensitivity of leaf explants of the fig cvs. BrownTurkey and Smyrna to kanamycin.

FIGS. 7 a-d show histochemical GUS analysis by stereomicroscopeobservations. (A) Transient GUS expression 3 days after inoculation ofleaf explants of Brown Turkey (0.63×1.0). (B) GUS staining ofregenerating leaf explants after four weeks culture in selection of 50mg/l kanamycin and 150 mg/l ticarcillin (0.63×2.5). (C) GUS expressiondetected in the leaves of isolated putatively transformed shoot of cv.Brown Turkey and (D) cv. Smyrna cultured on PM with 100 mg/l kanamycinand 150 mg/l ticarcillin (0.63×2.0).

FIGS. 8 a-b show PCR analysis of genomic DNA isolated from putativelytransgenic shoots of cv. Brown Turkey (A) and Smyrna (B). Thetransformed shoots showed the predicted bands of 645 bp for the npt IIgene and of 676 bp for the uidA-intron (GUS).

FIG. 9 presents Southern analysis of genomic DNA isolated fromputatively transformed shoot of fig cv. Brown Turkey and Smyrna. Thetransformed shoots showed the anticipated bands of the pME504 plasmidrestriction map.

FIG. 10 is a schematic illustration showing a map of the pME504construct, carrying the nptII, the uidA-intron genes and the bar geneunder the control of the CaMV 35S promoter.

FIG. 11 depict herbicide resistance in control and transgenic BrownTurkey cv treated with increasing concentrations of PPT.

FIG. 12 shows transgenic regeneration of ‘Brown Turkey’ fig cultivartransformed with pME504 and selection on 5 ppm BASTA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to genetically modified plants of thegenus Ficus, and parts thereof, transformed with a genetic constructcomprising at least one exogenous polynucleotide. The invention furtherrelates to methods of (i) transforming Ficus explants and (ii)regenerating the transformed explants to obtain transgenic Ficus plants.The invention is also relates to products produced by or from thetransgenic plants of the present invention. Specifically, the presentinvention relates to transgenic fig trees (Ficus carica), and to theiruse, inter alia, for the production of foreign proteins, as ediblevaccines and as improved stocks for agronomical use.

According to the present invention, the genome of commercially valuablewoody plants of the genus Ficus is modified by introducing at least oneexogenous polynucleotide, either homologous or heterologous to the Ficusgenome. The introduced polynucleotide can modify the production and/orfunction of a polypeptide of interest, for example increasing the amountof a rate-limiting enzyme by introducing additional copies of the gene.A reduction in the level of a polypeptide of interest can be achieved bytransforming the target plant with at least one antisense copy of a geneencoding the polypeptide, or a functional portion thereof. Non-codingportions of polynucleotides, such as a regulatory polynucleotide and apolynucleotides encoding regulatory factor such as transcription factor,and/or functional portions of a transcription factor, and/or antisensecopy of such a regulatory factor, can also be introduced to the targetFicus plants to modulate the expression of certain polypeptides. Anexogenous polypeptide can also be introduced for the production of aforeign protein.

According to certain embodiments of the present invention, theintroduced polynucleotide is expressed in a tissue specific manner toproduce the foreign protein. According to one embodiment, the foreignprotein is produced in an edible part of the plant, particularly in thefruit of Ficus carica. The fig tree is particularly advantageous as aspecies for production of foreign proteins due to the prolonged durationof ripening of the fruit on a single tree. Certain fig tree varieties,whether for use as a source of fresh fruit or dried fruit, have beendeveloped to have fruit which are ripening during the course of months,and even the year round. This provides unique advantages for the use offigs as a species suitable for use for fruit-specific production offoreign proteins, and as edible vaccines. Foreign proteins to beproduced by the transgenic fig tree are of nutritional and/orpharmaceutical value. As fig fruit are widely consumed, the geneticallymodified fruit can be used for administering nutritional and/orpharmaceutical compositions to animals and humans.

According to yet another embodiment, the foreign protein is expressed inthe latex serum produced by Ficus species. The foreign proteins producedby the transgenic fig trees may be isolated from the plant tissue, ormay be used while maintained within the plant tissue.

According to one aspect, the present invention provides transgenic Ficusspecies comprising a genetic construct comprising at least one exogenouspolynucleotide. According to one embodiment, the Ficus species is Ficuscarica.

The genetic material includes at least one exogenous polynucleotidedesired to be introduced to the target Ficus plant. Genetic constructsintroduced into the target plant may comprise a polynucleotide that ishomologous and/or heterologous to the target plant genome, and mayinclude polynucleotides encoding a polypeptide or a functional portionof a polypeptide, a polynucleotide encoding a regulatory factor, such asa transcription factor, a non-coding polynucleotides such as regulatorypolynucleotides, and antisense polynucleotides that inhibit expressionof a specified polypeptide. According to one embodiment, the geneticconstruct further comprises at least one regulatory element to conferfunctional expression of the exogenous polynucleotide in the Ficusplant. Expression of a polynucleotide refers to the process wherein aDNA region which is operably linked to appropriate regulatory element istranscribed into an RNA which is biologically active i.e., which iseither capable of interaction with another nucleic acid or which iscapable of being translated into a polypeptide or protein. As usedherein, the term “polypeptide” encompasses amino acid chains of anylength, including full-length proteins, wherein amino acid residues arelinked by covalent peptide bonds.

According to one embodiment, the genetic construct according to thepresent invention is built in such a way to further include at least onemarker gene conferring the ability to select transformed cells andtissues regenerated therefrom, for example callus, embryos and matureplants.

The transgenic Ficus plants of the present invention can be transformedwith any polynucleotide of interest. As used herein “polynucleotide”means a polymeric collection of nucleotides and includes DNA andcorresponding RNA molecules, both sense and anti-sense strands, andcomprehends cDNA, genomic DNA and recombinant DNA, as well as wholly orpartially synthesized polynucleotides. A polynucleotide may be an entiregene, or any portion thereof. Operable anti-sense polynucleotides maycomprise a fragment of the corresponding polynucleotide, and thedefinition of “polynucleotide” therefore includes all such operableanti-sense fragments. Identification of genomic DNA and heterologousspecies DNAs can be accomplished by standard DNA/DNA hybridizationtechniques, under appropriately stringent conditions, using all or partof a cDNA sequence as a probe to screen an appropriate library.Alternatively, PCR techniques using oligonucleotide primers that aredesigned based on known genomic DNA, cDNA and protein sequences can beused to amplify and identify genomic and cDNA sequences. Synthetic DNAscorresponding to the identified sequences and variants may be producedby conventional synthetic methods. All of the polynucleotides describedherein are isolated and purified, as those terms are commonly used inthe art.

When the genetic construct comprises a coding portion of apolynucleotide, the genetic construct further comprises a gene promotersequence and a gene termination sequence operably linked to thepolynucleotide to be transcribed. The gene promoter sequence isgenerally positioned at the 5′ end of the polynucleotide to betranscribed, and is employed to initiate transcription of thepolynucleotide. Promoter sequences are generally found in the 5′non-coding region of a gene but they may exist in introns or in thecoding region. When the construct includes an open reading frame in asense orientation, the gene promoter sequence also initiates translationof the open reading frame. For genetic constructs comprising either anopen reading frame in an antisense orientation or a non-coding region,the gene promoter sequence may comprise a transcription initiation sitehaving an RNA polymerase binding site.

A variety of gene promoter sequences that may be usefully employed inthe genetic constructs of the present invention are well known in theart. The promoter gene sequence, and also the gene termination sequence,may be endogenous to the Ficus host or may be exogenous, provided thepromoter is functional in species of the genus Ficus. For example, thepromoter and termination sequences may be from other plant species,plant viruses, bacterial plasmids and the like.

The type of the requested expression will dictate the selection of asuitable promoter. The promoters may be constitutive, inducible,tissue-specific, or developmentally regulated. Promoter hybrids can alsobe constructed to enhance transcriptional activity (e.g., U.S. Pat. No.5,106,739), or to combine desired transcriptional activity and tissuespecificity.

Promoters often used for constitutive gene expression in plants includethe CaMV 35S promoter, the enhanced CaMV 35S promoter, the FigwortMosaic Virus (FMV) promoter, the mannopine synthase (mas) promoter, thenopaline synthase (nos) promoter, and the octopine synthase (ocs)promoter. A suitable inducible promoter may be selected from genes thatare induced during a plant defense response against a parasiteinfection. For example, a fungal infection triggers an induction of alarge number of pathogenesis-related (PR) proteins by the infectedplant. The promoters of these PR sequences may be obtained and utilizedin the present invention. Isolation of these PR promoters has beenreported from potato plants (e.g., Matton, D. P. and Brisson, N. 1989.Mol Plant Microbe Interact. 2:325-31) and tobacco plants. Otherinducible promoters are heat-shock promoters, a nitrate-induciblepromoter derived from the spinach nitrite reductase sequence,hormone-inducible promoters, and light-inducible promoters associatedwith the small subunit of RuBP carboxylase and light harvestingchloroplast binding protein (LHCP) families.

According to one embodiment, the plant-expressible promoter is a tissuespecific promoter. Using tissue specific promoters restricts theexpression of the exogenous polynucleotide to the tissue where thepromoter is operable. According to one currently preferred embodiment,the tissue specific promoter is specific to any one of the tissuesforming the edible fig fruit.

The gene termination sequence, which is located 3′ to the DNA sequenceto be transcribed, may come from the same gene as the gene promotersequence or may be the termination sequence of a different gene. Manygene termination sequences known in the art may be usefully employed inthe present invention, such as the 3′ end of the Agrobacteriumtumefaciens nopaline synthase gene. However, preferred gene terminatorsequences are those from the original polypeptide gene, or from thetarget Ficus species being transformed.

The genetic construct of the present invention can further comprise areporter gene or a selection marker that is effective in the targetplant cells to permit the detection of transgenic cells, tissues orplants containing the genetic construct. Such selection markers andreporter genes, which are well known in the art, typically conferresistance to one or more toxins and encode for a detectable enzymaticactivity, respectively. The nptII gene, whose expression results inresistance to kanamycin or hygromycin antibiotics, which are generallytoxic to plant cells at a moderate concentration, can be used as aselection marker as exemplified herein below. Alternatively, thepresence of the desired construct in transgenic cells may be determinedby means of other techniques that are well known in the art, includingPCR, Southern and Western blots.

Techniques for operatively linking the components of the geneticconstructs used to transform target plant materials are well known inthe art and include the use of synthetic linkers containing one or morerestriction endonuclease sites as described, for example, by Maniatis etal., (Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989).

According to one embodiment, the transgenic Ficus plant of the presentinvention comprise a polynucleotide conferring a desirable agronomicaltrait selected from the group consisting of insect tolerance, diseaseresistance, herbicide tolerance, rooting ability, cold tolerance,drought tolerance, salinity tolerance, modified growth rate, modifiedfruit development, fruit with better appearance, fruit with bettertaste, fruit with better storage ability, fruit with better shelf life,and improved dried fruit.

As used herein, the term “storage ability” refers to the time period inwhich a fresh produce of a plant or parts thereof may be stored beforemarketing, under favorable conditions, without a significant decrease inits quality. The term “shelf life” as used herein refers to the timeperiod in which a fresh produce of a plant or parts thereof may bestored on a market shelf without a significant decrease in its quality.

According another embodiment, the transgenic Ficus plant comprises apolynucleotide encoding a foreign protein. As used herein, the term“foreign protein” refers to a polypeptide or a protein not naturallypresent in the Ficus species to be transformed. According to oneembodiment, the foreign proteins are expressed in an edible part of theplant, specifically in the edible fruit of Ficus carica. According toanother embodiment, foreign proteins can be extracted from thetransgenic Ficus plants.

The fig is particularly advantageous as a species for production offoreign proteins, specifically for the production of edible vaccines dueto the prolonged duration of ripening of the fruit on a single tree,which, in few varieties, is during the year round. Thus, geneticallymodified fig trees can provide a constant supply of nutritionallyvaluable fruit and edible vaccines.

There are many examples of valuable proteins that are useful inpharmaceutical and industrial applications. Often these molecules arerequired in large quantities and in partially or highly purifiedformulations to maintain product quality and performance. Plants are aninexpensive source of proteins, including recombinant proteins. Methodsfor obtaining constitutive high expression of foreign proteins by plantsand their extraction are known in the art (for example, U.S. Pat. Nos.6,392,121 and 6,617,435, respectively).

According to one embodiment, the Ficus plant is the fig tree Ficuscarica and the exogenous polynucleotide encodes a polypeptidecontributing to the nutritional composition of the Ficus carica ediblefruit.

According to another embodiment, the Ficus plant is the fig tree Ficuscarica and the exogenous polynucleotide encodes a protein contributingto the therapeutic value of the Ficus carica edible fruit. Preferably,the protein is selected from the group consisting of an anti-canceragent, an anti oxidant and a protein eliciting an immunogenic responsein a mammal.

According to one currently preferred embodiment, the present inventionprovides a transgenic Ficus carica plant producing fruit comprising atleast one exogenous polynucleotide expressing one of the groupconsisting of a polypeptide, a functional portion of a polypeptide, apeptide, a protein and a fusion protein eliciting immunogenic responsein a mammal, wherein the expression is at a level such that upon oraladministration to a mammal, an immunogenic response is observed, thusforming an edible vaccine.

Vaccines are administered to human and animal to induce their immunesystem to produce antibodies against viruses, bacteria and other typesof pathogenic microorganisms. Thus, vaccination prevents the occurrence,or reduces severity of a disease once the body is attacked by suchpathogens. Diseases like smallpox have been practically disappeared dueto the development of immunization program. However, many vaccines fordiseases as poliomyelitis, measles, mumps, rabies, foot and mouth, andhepatitis B are still too expensive for the lesser-developed countriesto provide to their large human and animal populations, as thesecountries do not have the monetary funds to immunize their populationswith currently available vaccines. The immunization cost includes notonly the cost of producing the vaccine but the further cost of theprofessional administration, particularly as some vaccines requiremultiple doses to maintain immunity. Therefore, often, the countriesthat need the vaccines the most can afford them the least. It has beenpreviously shown that plants can be transformed and efficiently expressimmunogenic protein in an amount sufficient for protein purification andvaccine production as well as for eliciting immunogenic response in amammal consuming the transgenic plant or parts thereof. Geneticconstructs for tissue specific expression are known in the art, thus theimmunogenic proteins can be expressed specifically in the plant edibleparts. Fig fruit, which are consumed for their nutritional value cantherefore provide inexpensive and highly accessible source for variousvaccines. Thus, the oral vaccines produced by the transgenic plants ofthe present invention are preferably administered by the consumption ofthe fruit produced by the transgenic plant. However, immunogeniccompositions derived from the transgenic plant materials suitable foruse as more traditional immune vaccines may be also readily preparedfrom the transgenic plant materials described herein. Preferably, suchimmune compositions will comprise a material purified from thetransgenic plant. Purification of the antigen may take many forms as isknown to those of skill in the art. Whatever initial purification schemeis utilized, the purified material will also be extensively dialyzed toremove undesired small molecular weight molecules (i.e., sugars,pyrogens) and optionally lyophilized for more ready formulation into adesired vehicle.

The vaccine can be any vaccine as is known to a person skilled in theart, including but not limited to a vaccine for the treatment ofhepatitis, malaria, and cholera.

Pollen and ovules from the transgenic Ficus plants; the seeds producedfrom same and the plants grown from the seeds; edible fruit produced bythe genetically modified plants; and plants regenerated from tissuecultures regenerated from the genetically modified plants of the presentinvention are also encompassed within the scope of the presentinvention.

According to yet another embodiment, the present invention provides atissue culture regenerated from the transgenic Ficus plants of thepresent invention, wherein the tissue culture comprises cells orprotoplasts from a tissue selected from the group consisting of leaves,pollen, embryos, roots, root tips, anthers, flowers, fruit and seeds.

According to another aspect, the present invention provides a method forproducing genetically modified Ficus species comprising:

a) transforming a genetic construct comprising at least one exogenouspolynucleotide into at least one Ficus explant to form a putativelytransformed explant;

b) inducing the formation of at least one adventitious shoot on theputatively transformed explant to obtain putatively transformedadventitious shoot;

c) selecting transformed adventitious shoot; and

d) culturing the transformed adventitious shoot to form a transgenicFicus plantlet.

According to one embodiment the methods of the present invention employin vitro fig shoot cultures as a starting material from which theexplants are obtained. Preparation of shoot cultures is known in the art(for example, Pontikis and Melas, 1986. Hort Science 21/1, pp. 153).Briefly, cultures are initiates from shoot tips. The elongated shootsare grown on a solid propagation medium until leaves are developed.According to one embodiment, the shoots are placed on a solidpropagation medium in a horizontal position in a Magenta box, withmultiple shoots per box. In addition to a basal salt mixture the mediumcomprises at least one auxin, at least one cytokinin and optionally atleast one gibberellin. Typically, the basal salt mixture used is fullstrength MS (Murashige and Skoog) medium, the auxin is indol-3-butyricacid (IBA), the cytokinin is 6-benzylaminopurine (BA) the gibberellicacid is GA₃. The medium typically further comprises sucrose as a carbonsource. The medium further comprises myo-inositol and thiamine-HCl andthe pH of the medium is kept in the range of from about 4.5 to about6.5.

The cultures are exposed to a cool fluorescent light in a photoperiod of16 h of light and 8 h dark, at 25° C. Typically, the light intensity isin the range of between 30 to 50 μmol/m² s. Under theses conditions (thepropagation medium and the light regime) elongation of themicropropagating shoots and the formation of leaves from the shoot budsoccur within about four to five weeks.

In the last two decades, transformation technology has played anincreasingly important role in the genetic manipulation of crop plantsfor their improvement and the study of the molecular mechanismsunderlying plant gene expression and regulation. However, due to thelack of a useable transformation and regeneration procedure, theapplication of such biotechnological approaches has not been possiblefor species in the genus Ficus. Successful transformation of Ficus cellsusing transformation procedures as described herein, and the subsequentregeneration of transgenic plants, provide a new mean for theintroduction of foreign genes into Ficus species, specifically into thecommercially valuable fig tree (Ficus carica). This technology enablesthe development of transgenic Ficus varieties with improved agronomicperformance characteristics, provides a new experimental system forstudy of gene expression and function in these species, and provides amean for the production of foreign proteins in edible parts of Ficusspecies, specifically the production of edible vaccines. The use ofFicus carica varieties improved via the utilization of thetransformation and regeneration technology of the present invention alsofacilitates the implementation of sustainable agricultural practices infig tree cultivation.

Methods for polynucleotide transfer into a plant cell are known in theart, and include, inter alia, Agrobacterium-mediated introduction,protoplast fusion, viral-mediated transformation, high velocityprojectile introduction, electroporation, injection into reproductiveorgans, and injection into immature embryos.

The probably most common method utilized for plants isAgrobacterium-mediated transformation. In addition, additional methodsdefined as “direct” gene transfer procedures have been developed totransform plants and plant tissues without the use of an Agrobacteriumintermediate. Plant regeneration from protoplasts is a particularlyuseful technique (Evans, D. A. et al., 1983. Handbook of Plant CellCulture 1, 124). When a plant species can be regenerated fromprotoplasts, direct gene transfer procedures can be utilized andtransformation is not dependent on the use of Agrobacterium. In thedirect transformation of protoplasts the uptake of exogenous geneticmaterial into a protoplast may be enhanced by use of a chemical agent(for example Polyethylene glycol) or electric field (electroporation).The exogenous material may then be integrated into the nuclear genome.Alternatively, genetically modified plants may be obtained by fusion oftwo distinct protoplasts carrying different genetic material followed byregeneration of the fused protoplast.

DNA viruses have been also used as gene vectors in plants. For example,a cauliflower mosaic virus carrying a modified bacterialmethotrexate-resistance gene was used to infect a plant, and the foreigngene was systematically spread in the plant. The advantages of thissystem are the ease of infection, systematic spread within the plant,and multiple copies of the gene per cell.

A recently developed procedure for direct gene transfer involvesbombardment of cells by microprojectiles carrying DNA (Klein, T. M. etal., 1987. Nature 327:70-72). In this “biolistic” procedure, tungsten orgold particles coated with the exogenous DNA are accelerated toward thetarget cells. Transient as well as stable expression has been achievedusing this procedure (Klein, T. M. et al., 1992. Bio/Technology 10:286-291).

According to one embodiment, transforming the genetic constructaccording to the present invention is performed by co-culturing the atleast one explant with Agrobacterium harboring the genetic constructcomprising the desired exogenous polynucleotide such that the geneticconstruct is transformed into the explant.

The term “Agrobacterium” refers to a soil-borne, Gram-negative,rod-shaped phytopathogenic bacterium, which causes crown gall. The term“Agrobacterium” includes, but is not limited to, the strainsAgrobacterium tumefaciens, (which typically causes crown gall ininfected plants). Infection of a plant cell with Agrobacterium generallyresults in the production of opines (e.g., nopaline, agropine, octopineetc.) by the infected cell. Thus, Agrobacterium strains which causeproduction of nopaline (e.g. strain LBA4301, C58, A208) are referred toas “nopaline-type” Agrobacteria; Agrobacterium strains which causeproduction of octopine (e.g. strain LBA4404, Ach5, B6) are referred toas “octopine-type” Agrobacteria; and Agrobacterium strains which causeproduction of agropine (e.g., strain EHA105, EHA101, A281) are referredto as “agropine-type” Agrobacteria. Any A. tumefaciens strain harboringa disarmed Ti plasmid may be used in the methods of the inventionutilizing any available Agrobacterium system may be used. For example,Ti plasmid/binary vector system or a co-integrative vector system withone Ti plasmid may be used. According to one embodiment, A. tumefaciensstrain EHA105 is used according to the methods of the present invention.

Colonies of Agrobacterium carrying the genetic construct of interest areprepared for inoculating the explants using methods which are well knownto one skilled in the art, as exemplified herein below. Explants takenfrom in vitro shoot cultures as described above are selected fortransformation. According to one embodiment, the explants are leadexplants. Before inoculating the explants with Agrobacterium theexplants are wounded; typically, when leaf explants are used, the leavesare wounded across the midrib. The terms “wounded” or “wounding” as usedherein refers to the introduction of a wound in the plant tissue.Wounding of plant tissue may be achieved, for example, by punching, byusing a blade, by maceration etc.

Inoculation of explants with the Agrobacterium suspension takes placeunder conditions that optimize infection of the explants. According toone embodiment, the explants are immersed in the Agrobacteriumsuspension for at least about twenty minutes, at a temperature of about22° C. to 30° C. After incubation, excess suspension is removed andexplants are transferred to a solid co-cultivation medium. The explantsare placed on the medium with their upper side facing the medium(abaxial side up). The present invention now discloses thattransformation is completely dependent on the orientation in which theexplants are placed on the medium during co-cultivation, i.e. notransformation occurs when the explants are placed in the oppositeorientation (adaxial side up).

According to another embodiment, the co-culturing medium comprises atleast one auxin and at least one cytokinin. According to one embodiment,the auxin is selected from indole-3-butyric acid (IBA) and2,4-dichlorophenoxy acetic acid (2,4-D) and the cytokinin is thidiazuron(N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, TDZ).

According to one embodiment, co-cultivation is performed for a period offrom about 65 h to about 80 h in the dark at a temperature range of 22°C.-28° C. After co-cultivation with Agrobacterium, putativelytransformed explants are transferred to a fresh medium to induce theformation of adventitious shoots and selection of transformed shoots.

Transformation of a cell may be stable or transient. The term “transienttransformation” or “transiently transformed” refers to the introductionof one or more exogenous polynucleotides into a cell in the absence ofintegration of the exogenous polynucleotide into the host cell's genome.Transient transformation may be detected by, for example, enzyme-linkedimmunosorbent assay (ELISA) which detects the presence of a polypeptideencoded by one or more of the exogenous polynucleotides. Alternatively,transient transformation may be detected by detecting the activity ofthe protein (e.g. β-glucuronidase) encoded by the exogenouspolynucleotide. The term “transient transformant” refers to a cell whichhas transiently incorporated one or more exogenous polynucleotides. Incontrast, the term “stable transformation” or “stably transformed”refers to the introduction and integration of one or more exogenouspolynucleotides into the genome of a cell. Stable transformation of acell may be detected by Southern blot hybridization of genomic DNA ofthe cell with nucleic acid sequences which are capable of binding to oneor more of the exogenous polynucleotides. Alternatively, stabletransformation of a cell may also be detected by enzyme activity of anintegrated gene in growing tissue or by the polymerase chain reaction ofgenomic DNA of the cell to amplify exogenous polynucleotide sequences.The term “stable transformant” refers to a cell which has stablyintegrated one or more exogenous polynucleotides into the genomic ororganellar DNA.

According to one embodiment, the explants removed from theco-cultivation medium are first washed. A preferred washing mediumcomprises MS medium comprising the anti-bacterial antibioticticaracillin, preferably at a concentration of from about 250 mg/l to350 mg/l, to eliminate the Agrobacterium. The putatively transformedexplants are then transferred to a fresh, solid regeneration mediumunder conditions which promotes bud regenerating and shoot formation.According to one embodiment, the regeneration medium further comprisesat least one selection agent. According to one currently preferredembodiment, the regeneration medium is based on MS medium comprising atleast one auxin, at least one cytokinin and at least one carbon source,further comprising at least two selection agents.

The success of gene transfer techniques is largely dependent onefficient regeneration system. Hitherto, only Yakushiji et al. (supra)have reported a successful regeneration of Ficus carica; however, aregeneration frequency of about only 22% obtained by the methodsdescribed therein is not sufficient for production of transgenic Ficusplants.

The present invention discloses a highly efficient system for in vitroregeneration of non-transformed as well as transformed species withinthe genus Ficus, specifically of fig tree (Ficus carica L.), showing forthe first time regeneration frequency of at least 80%, preferably 90%,more preferably 100%. Without wishing to be bound to a specificmechanism, a key factor in the high efficacy of both, transformation andshoot regeneration is the dorsoventral orientation of the explants,specifically leaf explants. In contrast to transformation, the highregeneration frequency is obtained when explants are put on the solidregeneration medium with their adaxial side up. When the explants areput with abaxial side up, although regeneration of shoots is observed,regeneration is apparent only on the free adaxial surface loosingcontact with the medium. The difference in the regeneration rate may bedue to the morphological differences between the adaxial and abaxialepidermal layers, requiring different threshold levels of hormones forregeneration. In addition, a regeneration efficacy of at least 3,preferably 5, more preferably 10 shoots per regenerating explant, isalso observed using the methods of the present invention.

According to one embodiment, the auxin in the regeneration medium is IBAat a concentration in the range of 1-3 ppm; the cytokinin is TDZ in aconcentration range of 1-3 ppm; and the carbon source is sucrose in aconcentration range of from about 2% to 6% (w/w). As exemplified hereinbelow, this combination of growth regulators and carbon source conferoptimal shoot regeneration from the transformed leaf explants of Ficuscarica. The putatively transformed explants are grown under low lightintensity for about one week and are then transferred to normal lightintensity. After a growth period of from about 3 weeks to about 10 weeksbud regeneration and adventitious shoot formation is observed. Duringthis growth period, the regenerating shoots are transferred to apropagation medium. According to one embodiment, the putativelytransformed explants are grown under low light intensity of from about 1to about 5 μmol/m² s and under normal light intensity of from about 10to about 60 μmol/m².

During the process of regeneration and development, transformedadventitious shoots containing the genetic construct are selected.

According to one embodiment the genetic construct of the presentinvention comprises a selection marker conferring antibiotic resistance.The transformation using a selection marker comprises:

a) exposing the putatively transformed explants to a regeneration mediumcomprising a first concentration of a first antibiotic and a secondantibiotic, to obtain surviving regenerated shoots;

b) transferring the surviving shoots to a propagation medium comprisinga second concentration of the first antibiotic and the secondantibiotic;

c) repeating step (b); and

d) transferring the surviving shoots of step (c) to the propagationmedium comprising the second antibiotic.

According to one embodiment, the regeneration and propagation media arethe media described above.

According to one embodiment, the second concentration of the firstantibiotic is greater than its first concentration, and theconcentration of the second antibiotic is constant. According to anothercurrently preferred embodiment, the first concentration of the firstantibiotic is 50 mg/l, the second concentration of the first antibioticis 100 mg/l and the concentration of the second antibiotic is 150 mg/l.According to one currently preferred embodiment, the first antibiotic iskanamycin and the second antibiotic is ticarcillin. This four-stageselection method substantially eliminates the presence of chimericshoots in the selected transformed shoots and prevents deleterious sideeffects of the antibiotics. When Agrobacterium-mediated transformationis employed, the presence of ticarcillin removes the remaining ofAgrobacterium.

Transformed shoots are transferred to a suitable rooting medium. Rootingmedia and methods of rooting are known to one skilled in the art.According to one embodiment, the root induction medium of the presentinvention comprises half concentration MS medium supplemented withmyo-inositol, thiamine HCl, phloroglucinol, and sucrose, at pH 5.7. Thetransformed shoots are transferred directly from theregeneration/selection medium to rooting cylinders with soil mixturemoistened with root induction medium, wherein the medium optionallycomprises 1 mg/l auxin, preferably IBA. Rooting is accomplished in aperiod of 10-30 days. According to one embodiment, plantlets are firstkept under high humidity conditions of 50-80% and are then transferredfor acclimatization and hardening by a stepwise decrease of the relativehumidity. However, the transformed shoots can generate healthy rootsystem when kept directly in ambient air conditions. Rooting efficacy(the percentage of transformed shoots which develop healthy root system)according to the present invention is at least 85%, preferably 95%, morepreferably 100%. Transformed plantlet may be grown to producegenetically modified mature plants.

According to one embodiment, the transformation and regeneration methodsof the present invention are utilized to introduce genetic material thatconfers desirable agronomical traits, including, but not limited toinsect tolerance, disease resistance, herbicide tolerance, rootingability, cold tolerance, drought tolerance, salinity tolerance,modification of growth rates and properties, fruit with betterappearance, fruit with better taste, fruit with better storage ability,fruit with better shelf life, and improved dried fruit. According tothis embodiment, the genetic material introduced may be homologous orheterologous to the genome of the target plant. Introducing new traitsto an existing plant variety by traditional breeding methods is a timeand work consuming process, even for herbaceous plants and moreover forwoody plants having a life cycle of years. In Ficus carica, using thetraditional genetic manipulation is even more complicated, as some treesbear only female synconium and others, named caprifig, bear both maleand female flowers. True fruits can be produced only on tress bearingfemale synconium only. In nature, cross-pollination is mediated by aspecific wasp. Thus traits donated by the stamens are hard to track andartificial pollination is hard to manipulate.

The origin of fig (Ficus carica, Moraceae) is tracked to Western Asia,from where it was spread to the Mediterranean. As of toady, fig treesare cultivated in many locations with an estimated annual production ofone million tons of fruit grown mainly in Turkey and Egypt and also inMorocco, Spain, Greece, California, Italy, Algeria, Syria and Tunisia.Approximately 40% of the fig crop is sold as dried fruit, with theremainder dividing between fresh produce, paste, juice and cannedpreserves. Dried and processed figs not suitable for human consumptioncan be used as animal fodder. The nutritional value of fresh figs iscomparable to that of many other fruits. They are high in calcium.

Dried figs, with only 20% water are nutritious relative to other freshfruits

There are two main commercial types of figs, the “common fig” thatproduces fruit without pollination, and the “Smyrna fig” that requirespollination by a fig wasp (Blastophaga spp.), that lives in the“caprifig” (male fig), to set fruit. The “common-type” (self-pollinated)fig is more commonly grown. These cultivars bear one or two crops peryear. The economic importance of fig production is likely to continueinto the future. Worldwide, there is an increasing demand for fresh figsand a stable demand for dried figs. However, the short storage and shelflife of fresh fruit, and lack of improved agricultural varieties limitsthe commercial growth of fig trees.

According to another embodiment, the transformation and regenerationmethods of the present invention are utilized to introduce heterologousgenetic material for the production of foreign proteins by Ficus carica.The foreign proteins to be produced by such genetically modified plantsare proteins that can modulate the nutritional composition and value ofthe edible fig fruit. According to one currently preferred embodiment,the introduced foreign protein is an anti-oxidant.

According to yet another embodiment, the transformation and regenerationmethods of the present invention are utilized to introduce heterologousgenetic material for the production of vaccines, particularly to theproduction of vaccines by the edible fruit of the fig trees.

According to another aspect, the present invention provides geneticallymodified Ficus species, specifically Ficus carica plants and partsthereof stably transformed with a genetic construct comprising at leastone polynucleotide of interest. Plants and parts thereof propagated fromthe genetically modified Ficus plants; Pollen and ovules from thegenetically modified Ficus plants; the seeds produced from same and theplants grown from the seeds; fruit produced by these plants; and plantsregenerated form tissue cultures regenerated from the plants of thepresent invention are also encompassed within the scope of the presentinvention.

According to yet another embodiment, the present invention provides atissue culture regenerated from the genetically modified Ficus plants ofthe present invention, wherein the tissue culture comprises cells orprotoplasts from a tissue selected from the group consisting of leaves,pollen, embryos, roots, root tips, anthers, flowers, fruit and seeds.

According to one embodiment, the genetically modified Ficus species areproduced by the transformation and regeneration methods of the presentinvention.

According to yet another aspect, the present invention provides agenetically modified Ficus plant wherein the plant is transformed with agenetic construct comprising polynucleotide encoding for at least oneprotein that is heterologous to the genome of the Ficus plant.

According to one embodiment, the present invention provides Ficus caricaplants stably transformed with a genetic construct comprisingpolynucleotide encoding for at least one polynucleotide, saidpolynucleotide encodes for a protein contributing to the nutritionalvalue of the Ficus carica edible fruit. According to one currentlypreferred embodiment, the protein is an anti-oxidant. According to onecurrently preferred embodiment, the protein is an anti-cancer agent.

According to another embodiment, the genetically modified plant of thepresent invention expresses at least one protein that can elicit animmunogenic response in a mammal. Preferably, the protein eliciting theimmunogenic response is expressed in an edible part of the plant,specifically in the fruit. According to one currently preferredembodiment, the present invention provides a genetically modified Ficuscarica plant producing fruit comprising protein eliciting immunogenicresponse in a mammal, wherein the protein is expressed in the fruit at alevel such that upon oral administration to an animal, an immunogenicresponse is observed, thus forming an edible vaccine.

The transformation and regeneration methods of the present invention canbe used to produce any type of vaccine effective in immunizing humansand animals against diseases. Viruses, bacteria, fungi, and parasitesthat cause diseases in humans and animals can contain antigenicprotein(s) which can confer immunity in a human or an animal to thecausative pathogen. A DNA sequence coding any of this viral, bacterial,fungal or parasitic antigenic protein may be used in the presentinvention. Mutant and variant forms of the DNA sequences encoding anantigenic protein which confers immunity to a particular virus,bacteria, fungus or parasite in a mammal (including humans) may also beutilized in this invention. For example, expression vectors may containDNA coding sequences which are altered so as to change one or more aminoacid residues in the antigenic protein expressed in the plant, therebyaltering the antigenicity of the expressed protein. Expression vectorscontaining a DNA sequence encoding only a portion of an antigenicprotein as either a smaller peptide or as a component of a new chimericfusion protein are also included in this invention.

As explained herein above, the production of the immunogenic protein isdirected to the fruit to produce edible vaccine, thus the geneticconstruct to be used for producing the vaccine comprises a fruitspecific promoter, operably linked to a polynucleotide encoding theimmunogenic protein. According to the present invention, anyAgrobacterium delivery system may be employed to transform Ficus caricawith the genetic construct.

The present invention allows for the production of not only a singlevaccine in the Ficus carica fruit but for a plurality of vaccines.Polynucleotides coding for multiple antigenic proteins can be includedin the genetic construct, thereby casing the expression of multipleantigenic amino acid sequences in one transgenic plant. Alternatively, aplant may be sequentially or simultaneously transformed with a series ofgenetic constructs, each of which contains DNA segments encoding one ormore antigenic proteins. For example, there are five or six differenttypes of influenza, each requiring a different vaccine. A transgenicplant expressing multiple antigenic protein sequences can simultaneouslyelicit an immune response to more than one of these strains, therebygiving disease immunity even though the most prevalent strain is notknown in advance.

The vaccine produced by the genetically modified Ficus plants accordingto the present invention can be any vaccine as is known to a personskilled in the art. According to one embodiment, the vaccine is selectedfrom the group consisting of a vaccine for hepatitis, malaria andcholera.

The principles of the invention, providing efficient transformation andregeneration methods for the production of genetically modified Ficuscarica plants according to the present invention, may be betterunderstood with reference to the following non-limiting examples.

EXAMPLES

Materials and Methods

Fig Shoot Culture Maintenance

Fig (Ficus carica L.) cultivars Brown Turkey (common for obtaining freshproduce) and Smyrna (common for dry fruits production) were used. Invitro shoot cultures of both cultivars were established according toPontikis and Melas (supra) and subsequently grown on proliferationmedium (PM) consisted of MS (Murashige and Skoog, 1962. Physiol. Plant.15, 473-497) basal salt mixture supplemented with 100 mg/l myo-inositol,0.5 mg/l thiamine-HCl, 3% sucrose (w/v), 0.8% agar (Sigma) and additionof 0.25 mg/l BA, 0.05 mg/l IBA and 0.05 mg/l GA₃. The pH was adjusted to5.7 prior to autoclaving for 20 min. at 120° C. Two shoots were placedhorizontally in Magenta boxes, containing 50 ml propagation medium andexposed to cool white fluorescent light (40 μmol/m² s) in a 16 hL/8 hDphotoperiod at 25° C. for 4-5 weeks before the leaf explants wereremoved.

Plant Regeneration

Regeneration experiments were carried out initially with the cultivarBrown Turkey. To examine the effect of culture media on adventitious budformation, the basal media MS, AP [13] and NN [14]; and variouscombinations of the auxins IBA and NAA (0.25-2 mg/l), NAA with thecytokinins TDZ (0.5-5 mg/l) and BA (2-5 mg/l) were examined. A controltreatment with only TDZ (0.5-5 mg/l) was performed. The regenerationmedia contained MS basal salt mixture, supplemented with 100 mg/lmyoinositol, 1 mg/l thiamine-HCl, 2-4% sucrose (w/v) and 0.8%, agar(Sigma), at pH 5.7. The effect of the addition of 0.25% activatedcharcoal (AC) was also examined.

The youngest three expanded leaves isolated from 3-4 weeks old plantswere used as explants. Each leaf was wounded forming three scaresperpendicular to the central vein and placed on regeneration medium withthe abaxial or adaxial surface down. Minimum ten Petri dishes, eachcontaining ten explants were used per treatment. The cultures were keptfor 7 days in low light intensity (2.5 μmol/m² s) followed by exposureto high light intensity (40 μmol/m² s) at 25° C., in a 16 hL/8 hDphotoperiod. Leaf explants were examined after 28 and 35 days and thepercentage of explant producing shoots (regeneration capacity) and themean of adventitious shoots formed per regenerating explant(regeneration efficacy) were recorded. All experiments were repeated atleast three times.

Rooting and Acclimatization

Two methods for rooting were examined. According to one method, eachshoot was cultivated individually in a tube with root induction (RI)medium composed of half concentration MS medium (½MS) supplemented with100 mg/l myo-inositol, 1 mg/l thiamine-HCl, 90 mg/l phloroglucinol, 2%sucrose (w/v), 0.25% activated charcoal (AC) and 0.8% agar (pH=5.7). Theeffect of IBA at different concentrations (0, 1, 2 mg/l) was alsotested. Each treatment included 32 individual plants. According toanother method, well developed shoots (up to 3-4 cm long with 3-4expanded leaves) were directly transplanted from the proliferationmedium to Rooting Cylinders (3.5 cm high×3.0 cm in diameter) with soilmixture comprising 55% granular polypropylene foam, 30% peat and 15%perlite (Tivonchem Ltd, Israel) moistened/soaked/with liquid RI mediumwithout AC, supplemented with either 0, 1, 2 mg/l IBA. Results werescored (0=shoot with no root; 1=rotting shoot) after 4 weeks and basedon 32 shoots (8 Magenta boxes with 4 cylinders) per treatment. Eachexperiment was repeated three times. Plantlets were cultured in closedboxes for one week in a growth chamber and then transferred for one weekto a greenhouse. Then the plantlets were further transferred to a highhumidity chamber without closures for additional 2 weeks for furtheracclimatization by decreasing the relative humidity stepwise.

Histology

The regeneration process was examined by light microscopy. Samples werefixed in a solution of freshly prepared FAA (Formal Acetic Alcohol),dehydrated in a graded ethanol series and embedded in Paraplast.Sections were cut at 10 mm thickness, stained in Safranin andFast-Green, mounted in Permount (Fisher) and examined, using Leica DMLBlight microscope.

Agrobacterium tumefaciens Strain and Plasmid

Supervirulent Agrobacterium tumefaciens strain EHA 105 (Hood et. al.,1993. Transgen. Res. 2, 208-218) harboring the vector pME 504 carryingthe nptII, and the uidA-intron genes (Vancanneyt et al., 1990. Mol. Gen.Genet. 220, 245-250) was used. Agrobacterium culture was grown overnightin LB medium (Duchefa L-1704) with appropriate antibiotics. Bacteriawere spun down by centrifugation (4000 rpm for 15 min), resuspended inliquid SIM medium (Alt-Möerbe et al., 1989. MPMI 2, 301-308)supplemented with 100 μM Acetosyringone (AS) to obtain a final OD₆₀₀ of0.7, and incubated in orbital shaker for 4 h at 28° C. and 250 rpm,After the incubation, the Agrobacterium culture was ready for use as aninoculum.

Transformation

The leaves of 3-4 weeks old micropropagated shoots were wounded acrossthe midrib with a scalpel and immersed in the bacterial suspension for20 min, blotted on filter paper and cultured on regeneration mediumbased on MS medium and supplemented with 2.0 mg/l TDZ and 2 mg/l IBA, 4%sucrose, 0.8% agar and pH 5.7. Co-cultivation medium was supplementedwith 100 μM AS. After co-cultivation period of 72 hours in the dark at25° C.±1 the explants were washed in liquid MS medium with 300 mg/lticarcillin, blotted dry and transferred to the regeneration medium withaddition of ticarcillin (150 mg/l) and kanamycin (50, 75 and 100 mg/l).The effect of paromomycin (25 and 50 mg/l) as selective agent also wastested.

In order to increase the efficiency of the selection a set of additionalexperiments was performed. Following co-cultivation with Agrobacteriuntumefaciens on solid medium the explants were cultured for 0, 3, 7 and10 days in liquid regeneration medium containing 50 mg/l kanamycin andticarcillin (150 mg/l). Ten leaves were placed in 10 ml liquid selectionmedium with addition of 0.5 ml Amberlite XAD-7 (Sigma) in Erlenmeyer(100 ml) in orbital shaker (85 rpm). Amberlite was sterilized byimmersing in 70% ethanol overnight, washed three times by sterile waterand mixed with equal portion (w/v) liquid selection medium. The liquidselection medium and amberlite were changed every 3 days. Then theexplants were blotted dry and transferred to solid selection medium with100 mg/l kanamycin and ticarcillin (150 mg/l) for the completion oftotal of four weeks. A control treatment with leaf explants transferreddirectly to solid medium with 100 mg/l kanamycin and 150 mg/lticarcillin was also performed. Each treatment was designed in fivereplications and the experiment was repeated twice.

All cultures were kept during the first week in low light intensity (2.5μmol/m² s) and subsequently transferred for the next 3 weeks tofluorescent light (40 μmol/m² s), in 16 hL/8 hD photoperiod at 25° C.±1.Regenerated shoots were developed after two subcultures in propagationmedium (PM) supplemented with 100 mg/l kanamycin and 150 mg/lticarcillin. Transformation frequency was counted as the number ofindependent transformation events (kanamycin resistant/GUS expressingshoots) obtained from the total number of explants.

Multiplication and maintenance of the selected plants were performed byculturing the shoots horizontally on PM with kanamycin (50 or 100 mg/l)and ticarcillin (150 mg/l) as individual clones. Rooting procedure wascarried out in rooting cylinders soaked with the liquid medium describedabove, containing 100 mg/l kanamycin. Acclimatization of the rootedplantlets to the greenhouse conditions was performed by progressivelydecreasing the relative humidity within 3-4 weeks.

Molecular Confirmation of Transformation

All selected clones were subjected to molecular analyses by PCR andSouthern blotting for the presence and integration of the nptII and GUSgenes. Plant genomic DNA was isolated from the youngest three leavesexcised from kanamycin resistant shoots according to Murray and Tompson(1980. Nucl Acids Res 8, 4321-4325).

The oligonucleotide primers used for the PCR amplification of a 645 bpfragment of the nptII gene were: 221924-Direct primer 5′-GCC GCT TGG GTGGAG AGG CTA T-3′ (SEQ ID NO:1) (63.6° C.); and 221925-Reverse primer5′-GAG GAA GCG GTC AGC CCA TTC-3′ (SEQ ID NO:2) (60° C.).

The primers for a 676 bp fragment of the GUS gene were: 17081-001-GUSup5′-CGA GCG ATT TGG TCA TGT GAA G-3′ (SEQ ID NO:3) (57.5° C.); and17081-002-GUSlow primer 5′-CAT TGT TTG CCT CCC TGC TGC GGT T- (SEQ IDNO:4) 3′ (55.9° C.) (Sigma).

Amplification was performed in aliquots of 25 μl using a thermal cycler(Biometra). The PCR conditions for amplification of the nptII genefragment were 95° C. for 5 min, followed by 35 cycles at 94° C. for 1min, 62° C. for 1 min, 72° C. for 1 min, and a final extension at 72° C.for 10 min. Amplification of the uidA-intron fragment was performed withthe following program: 95° C. for 5 min followed by 35 cycles at 94° C.for 45 s, 55° C. for 45 s, 72° C. for 45 s, and a final extension at 72°C. for 10 min.

For the Southern blot analysis, 10 μg of genomic DNA was digested withthe enzyme HindIII, subjected to electrophoresis on 0.8% agarose gel andtransferred to nylon membranes (GeneScreen Plus, NEN, Boston, Mass.).Southern hybridization was performed using a [P]-radioactively labeled524-bp nptII gene fragment as a probe, as described The membrane washedat high-stringency conditions in a buffer consisting of 0.1×SSC, and0.5% SDS at 65° C.

Histochemical GUS Assay

Histochemical analysis was performed following the procedure ofJefferson et al. (1987. EMBO J. 6, 3901-3907).

Transient GUS expression was assayed four and seven days afterinfection. Regenerating leaf explants cultivated four weeks in selectiveconditions of kanamycin (50 mg/l) and ticarcillin (150 mg/l) were alsotested for β-glucuronidase activity. GUS expression was also examined inputatively transformed shoots and in rooted plantlet selectiveconditions.

Example 1 Plant Regeneration

Effect of the Growth Regulators

Our preliminary experiments have shown that use of MS basal mediumsupplemented with a combination of IBA and TDZ resulted in shootformation rate of up to 28-30%, while supplementing the MS medium with acombination of NAA and TDZ resulted in a maximum regeneration of only upto 15%. Based on these observations, the medium used for theregeneration assays was MS medium comprising different concentrations ofIBA and TDZ. FIG. 1A-B shows the effect of growth regulatorsconcentration and two different light conditions on the regeneration ofleaf explants obtained from shoot grown in PM medium. Higherregeneration frequency was obtained when leaf explants were cultured forone week in low light intensity (2.5 μmol/m² s) and subsequentlytransferred to normal light intensity (40 μmol/m² s). A combination of 2mg/l IBA and 2 mg/l TDZ was shown to give the best regenerationfrequency of 50.2% with an average of 3.2±0.6 shoots per regeneratingexplant (FIG. 1A). Sufficient regeneration frequencies (42.6-46.6%) werealso obtained when the leaf explants were grown in a medium containing 1mg/l IBA and 2 mg/l TDZ; however, most of the explants turned brown andformed excessive calli. Culturing of control explants with TDZ onlyinduced significant explants expansion and compact calli formationhowever with a very low regeneration frequency of ca. 2%. The additionof AC did not positively influenced regeneration. Application of twoweeks dark treatment before the exposure of the plants to low lightintensity also was not advantage (FIG. 1B).

Hence, cultivating the leaf explants on MS basal medium supplementedwith a combination of 2 mg/l IBA and 2 mg/l TDZ, in light conditions ofone week low light intensity followed by transfer to normal light wasidentified as the best conditions for obtaining high regenerationfrequencies and increasing the percentage of shoot differentiation.

Histological observations (FIG. 2 A-C) showed that the regenerationprocess followed the developmental pattern of direct organogenesis.Adventitious shoots developed from meristematic centers, appearing inboth epidermal and mesophyll tissues. Within 25 days they became visibleon the wounded surface of the explant. A prolonged culture up to 42 daysresulted in explant expansion, but the developing shoots were surroundedand covered by significant amount of callus, which inhibited theirfurther growth.

Effect of the Leaf Explants Position on the Solid Medium and the MediumSucrose Concentration

The present invention surprisingly shows that the position of the leafsurface on the medium, in combination with the sucrose concentration inthe medium have a significant effect of the leaf explants regeneration.Data presented in FIG. 3 show that cultivating the explants with theiradaxial surface up led to a considerable increase in their regenerationfrequency. In combination with 4% sucrose, leaf explants cultured withtheir adaxial side up showed 100% of adventitious shoot formation withmore than 5 shoots per regenerating explant. Most of the adventitiousshoots developed directly, typically at the wounds sited at the centraland distal part of the leaf petiole (FIG. 4A). When the explants werecultured with their abaxial side up, shoot formation occurred on theadaxial side of the leaf only (FIG. 4B), and browning and calliformation were observed.

In conclusion, based on the results described herein above, the optimalconditions for fig regeneration from leaf explants are as follows: leafexplants are isolated from 3-4 weeks old in vitro fig shoot cultures;the explants are placed with their adaxial side up on an MS-basedregeneration medium containing 2 mg/l IBA, 2 mg/l TDZ and 4% (w/v)sucrose; explants cultures are exposed to low light intensity (2.5μmol/m² s) for one week and subsequently transferred to normal light (40μmol/m² s). Accumulation of the factors that positively influencedregeneration, enable us to develop an efficient and reproducible systemfor adventitious shoot formation in. This protocol was successfullyapplied to fig cultivar Brown Turkey and cv. Smyrna and resulted in 100%regeneration with more than 5 shoots per regenerating explant. Theregeneration system described herein above was used as a prerequisitefor the development of an efficient transformation system.

Rooting Procedure

Two methods were examined to obtained root induction (Table 1).According to the first approach, each regenerated shoot was placed in atube containing root induction medium comprising 2 mg/l IBA. Using thismethod, ca. 80% of the shoots formed roots. However, insufficientpercentage of these plants was successfully acclimatized in thegreenhouse conditions. According to the second approach, regeneratedshoots were cultured directly in rooting cylinders, with or without 1mg/l IBA. After 4 weeks showed 100% root formation was achieved,independent on the presence of an auxin. These plants had betterhardening and growth characteristics and easier further acclimatizationin the greenhouse conditions (FIG. 5). TABLE 1 Comparison of Methods forRoot Formation Root formation (%) ± SE IBA (mg/l) Rooting in liquidmedium Rooting in solid medium 0 8.3 ± 1.59 100 1  47 ± 5.43 100 2 81.3± 7.61  83 ± 6.27

Example 2 Transformation of Ficus carica

Choice of Selective Antibiotic

In order to use kanamycin as a selection agent for transformed cells,non-inoculated leaf explants from in vitro fig cultures of bothcultivars Brown Turkey and Smyrna were tested for their tolerance tokanamycin. Wounded leaves were placed on a regeneration mediumsupplemented with kanamycin at a concentration selected from 0, 10, 25,50, 75 and 100 mg/l. The minimal concentration for inhibition ofadventitious shoot induction for cv. Brown Turkey was 25 mg/l. Howeverwith cv. Smyrna, a few single buds were formed at 25 mg/l kanamycin, butthey remained white and did not develop further (FIG. 6 Km sensitivity).Therefore, the concentration of 50 mg/l kanamycin was initially chosenfor selection in the subsequent transformation experiments.

Regeneration of Transgenic Shoots

Leaf explants of 3-4 weeks old in vitro propagated Ficus carica plantswere wounded across the main vein and co-cultivated with the disarmedstrain EHA105 harboring the plasmid pME504, comprising the genes GUS-intand npt II. Following co-cultivation with A. tumefaciens, explants wereinitially placed on regeneration medium supplemented with 50 mg/lkanamycin and 150 mg/l ticarcillin and cultured with their adaxial sideup. The leaf explants were exposed for one week to low light intensityand then transferred to normal light. Under the selective conditions of50 mg/l kanamycin, up to 30% of the explants of Brown Turkey and up to50% of Smyrna formed green shoots after 4 weeks of culture. Theregenerating shoots were transferred to PR medium supplemented with 100mg/l kanamycin and 150 mg/l ticarcillin for further selection. Followingthis scheme of selection the transformation frequency established forcv. Brown Turkey varied between the experiments from 1.7 to 10% and from2.1 to 7.8% for cv. Smyrna. Our observations showed that cultivation ofthe new emerging transgenic shoots more than 3 subcultures in selectiveconditions of 100 mg/l kanamycin on proliferation medium resulted ininhibition of their growth and development. Therefore, the followingdesign of regeneration/selection process was concluded: one subculture(4 weeks) on regeneration medium with 50 mg/l kanamycin and 150 mg/lticarcillin followed by two subcultures (6 weeks) of the isolated shootson PM with 100 mg/l kanamycin and 150 mg/l ticarcillin and finally onesubculture only with ticarcillin (150 mg/l). Then leaves from theputative transgenic plants were subjected to GUS staining. GUS positiveplants were grown as individual clones on PM with 50 mg/l kanamycin withor without ticarcillin (150 mg/l) and subjected for molecular analysis.

In order to increase the efficiency of the selection a set of additionalexperiments was performed. Following co-cultivation with Agrobacteriumtumefaciens on solid medium the explants were cultured for 0, 3, 7 and10 days in liquid regeneration medium containing 50 mg/l kanamycin andticarcillin (150 mg/l). Leaves were placed in 10 ml liquid selectionmedium with addition of 0.5 ml Amberlite XAD-7 (Sigma) in Erlenmeyer(100 ml) in orbital shaker (85 rpm). The liquid selection medium andamberlite were changed every 3 days. Then the explants were blotted dryand transferred to solid selection medium with 100 mg/l kanamycin andticarcillin (150 mg/l) for the completion of total of four weeks.Following this scheme of selection the transformation frequencyestablished for cv. Brown Turkey varied between the experiments from 6.to 12% for cv. Smyrna it was from 4 to 10% respectively.

Histochemical GUS Assay

Transient GUS expression reveals a high gene transfer rate only forleaves that have been blotted during the co cultivation period withtheir adaxial side up. Histochemical GUS analysis done 3 and 7 daysafter infection showed that between 95 and 100% of the explants of bothcultivars had large infected sites, when cultured with their adaxialside up. However when cultured with their abaxial side up no transientGUS expression was observed in the two fig varieties examined (FIG. 7 A)After four weeks in selective conditions (kanamycin 50 mg/l) part of theregenerating explants (20 leaves) stained histochemically and positiveGUS shoots were counted. Observation under stereomicroscope (FIG. 7 B)of the leaves of cv. Brown Turkey showed that 7.46% (10 out of 134) ofthe shoots were GUS positive and 92.53% escaped transformation. An insitu β-glucuronidase assay was carried out to further confirm theintegration and expression pattern of the transformed marker genes.Intact plants of all the selected shoots propagated on a mediumcontaining kanamycin and control (wild type) plants were tested. GUSexpression was observed in the leaves of the transgenic fig plants cv.Brown Turkey (FIG. 7 C) and Smyrna (FIG. 7C).

Molecular Confirmation of Transformation

PCR analysis of the putative transgenic shoots confirmed the stableincorporation of the transgenes into the Ficus carica genome. All clonesselected after transformation with pME504 showed the predicted bands—the645 bp for the npt II gene and the 676 bp for the uidA-intron (GUS)(FIG. 8 A-B). No fragment was amplified in the control, untransformedplant (not shown).

Southern blot analysis on HindIII digested genomic DNA from one putativetransgenic plants of Brown Turkey and one putative transgenic plants ofSmyrna, randomly chosen, provided additional molecular evidence to theincorporation of foreign DNA (FIG. 9). The Southern blot data areconsistent with the plasmid (pME504) restriction map. No hybridizationbands were present in the control, untransformed plants (not shown).Southern blot analysis on Hind III digested DNA from the putativetransgenic plant provided additional evidence for the incorporation ofthe foreign DNA. The hybridization pattern in Smyrna indicated insertionof the T-DNA into two different loci (FIG. 9).

Example 3 Transgenic ‘Brown Turkey’ Plants Generated According to theTeachings of the Present Invention Expressing the Bar HerbicideSelective Marker

Bialaphos, a non-selective herbicide, is a tripeptide composed of twoL-Ala residues and an analog of Glu known as phosphinothricin (PPT).Bialaphos is toxic to bacteria and plants after intracellular peptidasesremove the Ala residues and release active PPT, an inhibitor of Glnsynthetase (GS). Inhibition of GS by PPT causes a rapid buildup ofintracellular ammonia levels. The associated disruption of chloroplaststructure results in inhibition of photosynthesis and plant cell death.There is thus a widely recognized need for generating plants resistantto PPT, Basta.

Transgenic ‘Brown Turkey’ plants expressing the bar gene, were generatedusing the teachings of the present invention. Such plants are expectedto display resistance to the herbicide phosphinothricin (PPT, ‘Basta’).

Experimental Procedures and Results

The pME504 plasmid which comprises the bar gene [Streptomyceshygroscopicus bar gene conferring resistance to herbicide bialaphos(X05822) Thompson, C. J., Movva, N. R., Tizard, R., Crameri, R., Davies,J. E., Lauwereys, M. and Botterman, J. Characterization of theherbicide-resistance gene bar from Streptomyces hygroscopicus EMBO J. 6,2519-2523 (1987)] was used to transform ‘Brown Turkey’ plants. Herbicideresistance in control and transgenic Brawn Turkey cv. tissue cultureline 001 was evaluated. As shown in FIG. 11, non-transformed plantsshowed signs of necrosis and chlorosis on basal leaves following twodays and died one week later (FIG. 11). Resistant lines, however, werepropagated in vitro even on 10 ppm Basta (FIG. 11).

These results demonstrate that the heterologous expression of the bargene in fig is associated with resistance to the herbicidephosphinothricin.

The possibility of the use of the bar gene as a selectable marker intransgenic experiments was also evaluated.

Transformation of ‘Brown Turkey’ fig cultivar with pME504, as above, andselection on 5 ppm BASTA yielded seven plants with transformationfrequency of 3.5% (FIG. 12).

These result demonstrate the feasibility of using the bar gene as aselectable marker that could replace the antibiotic resistance gene infig.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed chemical structures andfunctions may take a variety of alternative forms without departing fromthe invention.

1. A transgenic Ficus plant and parts thereof comprising a geneticconstruct comprising at least one exogenous polynucleotide.
 2. The plantof claim 1, wherein the genetic construct further comprises at least oneregulatory element.
 3. The plant of claim 2, wherein the regulatoryelement is selected from the group consisting of a promoter, anenhancer, a terminator, a transposable element and apost-transcriptional element.
 4. The plant of claim 1, wherein thegenetic construct further comprises a selectable marker.
 5. The plant ofclaim 4, wherein the selectable marker is a gene inducing antibioticresistance or a gene inducing herbicide resistance.
 6. The plant ofclaim 4, wherein the selectable marker is a reporter gene coding for adetectable protein.
 7. The plant of claim 1, wherein the exogenouspolynucleotide is selected from the group consisting of a polynucleotidehomologous to the Ficus genome and a polynucleotide heterologous to theFicus genome.
 8. The plant of claim 7, wherein the exogenouspolynucleotide is selected from the group consisting of a polynucleotideencoding a polypeptide, a polynucleotide encoding a functional portionof a polypeptide, a polynucleotide encoding a regulatory factor, anon-coding regulatory polynucleotide, and an antisense polynucleotide.9. The plant of claim 8, wherein the exogenous polynucleotide confer adesirable agronomical trait selected from the group consisting of insecttolerance, disease resistance, herbicide tolerance, rooting ability,cold tolerance, drought tolerance, salinity tolerance, modified growthrate, modified fruit development, fruit with better appearance, fruitwith better taste, fruit with better storage ability, fruit with bettershelf life, and improved dried fruit.
 10. The plant of claim 8, whereinthe exogenous polypeptide encodes a foreign protein.
 11. The plant ofclaim 10 wherein the foreign protein is expressed in the latex serumproduced by the plant.
 12. The plant of claim 10, wherein the foreignprotein is expressed in an edible part of the plant.
 13. The plant ofclaim 12, wherein the foreign protein contributes to the nutritionalcomposition of the Ficus edible part.
 14. The plant of claim 12, whereinthe foreign protein contributes to the therapeutic value of the Ficusedible part.
 15. The plant of claim 14, wherein the foreign protein isselected from the group consisting of an anti-cancer agent, an antioxidant and a protein eliciting an immunogenic response in a mammal. 16.The plant of claim 15 wherein the foreign protein is a protein elicitingan immunogenic response in a mammal.
 17. The plant of claim 16 whereinthe immunogenic response is observed in the mammal upon oraladministration of the edible part thus forming an edible vaccine. 18.The plant of claim 17, wherein the vaccine is for the treatment of adisease selected from the group consisting of hepatitis, malaria andcholera.
 19. The plant of claim 1, wherein the transgenic Ficus is Ficuscarica.
 20. A transgenic Ficus carica plant producing fruit comprisingat least one exogenous polynucleotide expressing one of the groupconsisting of a polypeptide, a functional portion of a polypeptide, apeptide, a protein and a fusion protein eliciting immunogenic responsein a mammal, wherein the expression is at a level such that upon oraladministration to a mammal, an immunogenic response is observed.
 21. Amethod for producing transgenic Ficus species comprising: (a)transforming a genetic construct comprising at least one exogenouspolynucleotide into at least one Ficus explant to form a putativelytransformed explant; (b) inducing the regeneration of at least oneadventitious shoot on the putatively transformed explant to obtainputatively transformed adventitious shoot; (c) selecting a transformedadventitious shoot; and (d) culturing the transformed adventitious shootto form a transgenic Ficus plantlets.
 22. The method of claim 21 whereinthe exaplant is obtained from an in vitro Ficus culture.
 23. The methodof claim 22, wherein the in vitro culture is prepared by horizontallyplacing shoot tips of Ficus on a solid propagation medium enabling shootgrowth and leaf development.
 24. The method of claim 23 wherein thepropagation medium comprises at least one cytokinin, at least one auxinand optionally at least one gibberellin.
 25. The method of claim 21,wherein the explant is leaf explant.
 26. The method of claim 21, whereintransforming the explant comprises co-culturing the explant withAgrobacterium culture harboring the genetic construct.
 27. The method ofclaim 26, wherein co-culturing is performed with explant placed on asolid medium, abaxial side up.
 28. The method of claim 27, wherein theexplant is wounded before culturing with Agrobacterium.
 29. The methodof claim 26, wherein the Agrobacterium is A. tumefaciens.
 30. The methodof claim 27, wherein the solid culturing medium comprises at least oneauxin and at least one cytokinin.
 31. The method of claim 21, whereininducing the formation of an adventitious shoot is obtained by placingthe putatively transformed explant, adaxial side up, on a solidregeneration medium under low light intensity; transferring the explantto normal light intensity; and subsequently transferring the explant toa propagation medium under normal light intensity.
 32. The method ofclaim 31, wherein the low light intensity is in the range of 1-5 μmol/m²s.
 33. The method of claim 31, wherein the normal light intensity is inthe range of 10-60 μmol/m² s.
 34. The method of claim 31, wherein theregeneration medium comprises at least one auxin, at least one cytokininand at least one carbon source.
 35. The method of claim 34, wherein theregeneration medium further comprises at least one selection agent. 36.The method of claim 35, wherein the selection agent is an antibiotic.37. The method of claim 21, wherein the genetic construct furthercomprises a selection marker.
 38. The method of claim 37, wherein theselection marker is a gene inducing antibiotic resistance or a geneinducing herbicide resistance.
 39. The method of claim 38, whereinselecting the transformed shoot is performed using a selection methodcomprising: (a) exposing the putatively transformed explants to aregeneration medium comprising a first concentration of a firstantibiotic and a second antibiotic, to obtain surviving regeneratedshoots; (b) transferring the surviving shoots to a propagation mediumcomprising a second concentration of the first antibiotic and the secondantibiotic; (c) repeating step (b); and (d) transferring the survivingshoots of step (c) to the propagation medium comprising the secondantibiotic.
 40. The method of claim 39 wherein the second concentrationof the first antibiotic is greater than its first concentration, and theconcentration of the second antibiotic is constant.
 41. The method ofclaim 39, wherein the first antibiotic is kanamycin and the secondantibiotic is ticarcillin.
 42. The method of claim 31 wherein thefrequency of shoot regeneration is at least 80%, preferably 90%, morepreferably 100%.
 43. The method of claim 21, wherein the geneticconstruct further comprises at least one regulatory element.
 44. Themethod of claim 43, wherein the regulatory element is selected from thegroup consisting of a promoter, an enhancer, a terminator, atransposable element and a post-transcriptional element.
 45. The methodof claim 21, wherein the exogenous polynucleotide is selected from thegroup consisting of a polynucleotide homologous to the Ficus genome anda polynucleotide heterologous to the Ficus genome.
 46. The method ofclaim 45, wherein the exogenous polynucleotide is selected from thegroup consisting of a polynucleotide encoding a polypeptide, apolynucleotide encoding a functional portion of a polypeptide, apolynucleotide encoding a regulatory factor, a non-coding regulatorypolynucleotide, and an antisense polynucleotide.
 47. The method of claim45, wherein the exogenous polynucleotide confer a desirable agronomicaltrait selected from the group consisting of insect tolerance, diseaseresistance, herbicide tolerance, rooting ability, cold tolerance,drought tolerance, salinity tolerance, modified growth rate, modifiedfruit development, fruit with better appearance, fruit with bettertaste, fruit with better storage ability, fruit with better shelf life,and improved dried fruit.
 48. The method of claim 45, wherein theexogenous polypeptide encodes a foreign protein.
 49. The method of claim48, wherein the foreign protein is expressed in the latex serum producedby the plant.
 50. The method of claim 48, wherein the foreign protein isexpressed in an edible part of the plant.
 51. The method of claim 50,wherein the foreign protein contributes to the nutritional compositionof the Ficus edible part.
 52. The method of claim 50, wherein theforeign protein contributes to the therapeutic value of the Ficus ediblepart.
 53. The method of claim 52, wherein the foreign protein isselected from the group consisting of an anti-cancer agent, an antioxidant and a protein eliciting an immunogenic response in a mammal. 54.The method of claim 53, wherein the foreign protein is a proteineliciting an immunogenic response in a mammal.
 55. The method of claim54 wherein the immunogenic response is observed in the mammal upon oraladministration of the edible part thus forms an edible vaccine.
 56. Themethod of claim 55, wherein the vaccine is for the treatment of adisease selected from the group consisting of hepatitis, malaria andcholera.
 57. The method of claim 20, wherein the transgenic Ficus isFicus carica.